0021-972X/97/$03.00/0 Vol. 82, No.

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Journal of Clinical Endocrinology and Metabolism Printed in U.S.A.
Copyright © 1997 by The Endocrine Society

CLINICAL REVIEW 87
In Vitro Fertilization for Male Factor Infertility
PETER N. SCHLEGEL AND SARAH K. GIRARDI
James Buchanan Brady Foundation, Department of Urology, The New York Hospital-Cornell Medical Center, and
The Population Council, Center for Biomedical Research, New York, New York

M ALE FACTOR infertility is a general term that de-scribes

couples in whom an inability to conceive is associated with a
problem identified in the male partner. This problem may be
associated with low sperm production (oli-gospermia), poor
sperm motility (asthenospermia), or ab-normal morphology
(teratospermia) (1). Abnormal sperm function may also be
evaluated with sperm function tests that evaluate sperm
interaction with cervical mucus (cervical mu-cus penetration test),
the zona pellucida surrounding the oocyte (hemi-zona binding
assay), or the oocyte itself (ham-ster-egg penetration assay) (2).
Male factor infertility also describes men with normal sperm
production but conditions that prevent sperm transport to the
vagina during inter-course (e.g. reproductive tract obstruction or
ejaculatory dys-function). Obtaining serial semen analyses and
questioning of the male partner should be part of the initial survey
of an infertile couple. When a male factor is suspected during
evaluation of a couple for infertility, complete evaluation of the
man is warranted. If treatable conditions causing the male factor
are found, they should be corrected. If treatment is unsuccessful,
or if the couple still does not conceive, then assisted reproduction
is indicated. Assisted reproductive techniques include intrauterine
insemination (IUI), in vitro fertilization (IVF), and IVF with
micromanipulation. Micro-manipulation refers to a series of
procedures that enhance the ability of sperm to fertilize an oocyte,
in vitro. In this review we will emphasize recent advances in IVF,
especially IVF with the advanced micromanipulation technique of
intracy-toplasmic sperm injection (ICSI), as tools for treatment of
the
infertile couple with male factor infertility.
Evaluation of male factor infertility
The cornerstones of evaluation of a subfertile man include a
comprehensive history, physical examination, multiple se-men
analyses, and an endocrine evaluation. In specific cir-cumstances,
additional testing may be indicated. For men with azoospermia or
severe oligospermia (sperm concentra-tion , 5 3 106/cc),
consideration of karyotypic abnormalities such as Klinefelter's
syndrome is appropriate if clinically
Received March 1, 1996. Revision received July 11, 1996. Re-revision
received October 18, 1996. Accepted October 28, 1996.
Address correspondence and requests for reprints to: Peter N. Schlegel,
M.D., Room F-905A, Department of Urology, The New York Hospital-
Cornell Medical Center, 525 East 68th Street, New York, New York 10021.
indicated. In addition, up to 13% of men with azoospermia may
have microdeletions of the Y chromosome. Although routine
evaluation for these microdeletions is available at only a few U.S.
academic centers in 1996, evaluation at the SIMMY protocol
referral center (Study of ICSI, Male Infer-tility and
Microdeletions on the Y chromosome) can provide free testing of
patients who are candidates for treatment with ICSI (3± 6). For
men with unilateral or bilateral congenital absence of the vas
deferens, cystic fibrosis transmembrane conductance regulator
(CFTR) gene analysis is important, as 55± 82% of men with
congenital absence of the vas deferens will carry detectable CFTR
mutations (7). In addition, pa-tients with idiopathic epididymal
obstruction have been es-timated to have a 47% chance of
carrying a detectable CFTR mutation (8). For couples with vasal
or epididymal anoma-lies, testing of the female partner for CFTR
mutations is even more important, as not all CFTR mutations are
currently detectable in the man. In the case of any other genetic
con-dition, including treatment of men with Klinefelter's syn-
drome, and in the case of couples with a female partner over age
40 yr, genetic counselling is recommended before as-sisted
reproduction treatments.
Treatment of male infertility
Up to 75% of men with a male factor will have identifiable or
treatable conditions that affect their fertility (9 ±11). Nearly all
men with male factor infertility are treatable with assisted
reproductive techniques. Before applying more invasive
techniques, however, avoidance of specific gonadotoxic fac-tors
such as exogenous heat, chemical gonadotoxins (e.g.
sulfasalazine and cimetidine), or medications that can ad-versely
affect fertilization (including calcium channel block-ers) is
appropriate. Treatment of varicoceles, endocrine disturbances,
symptomatic infections, and obstructive azoospermia have all
been demonstrated, using randomized or other appropriately
designed studies, to have a role in the management of male
infertility (12). Specific treatment of the man may be less
invasive, more successful, and more cost effective (13, 14) with
lower risk than IVF. In addition, it is worthwhile to remember
that up to 1% of men with subfer-tility have a potentially life
threatening condition associated with their fertility problem, (e.g.
testis tumor) (1, 15). Suffice it to say that evaluation and
treatment of a man with male factor is worthwhile, despite the
recent advances in assisted reproduction.

709

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710
Background: in vitro fertilization
Male factor infertility was initially considered a contrain-
dication to IVF because abnormal sperm are less likely to fertilize
oocytes than normal sperm (16). However, subse-quent
experience starting just over a decade ago indicated that
fertilizations and subsequent live births were possible despite
impaired sperm quality (17). IVF has had some suc-cess in the
treatment of these men, however it has been recognized that even
normal concentrations of sperm from oligozoospermic men,
placed directly with oocytes in cul-ture, do not fertilize at the
same rates as sperm from other-wise normal men. In addition,
adequate numbers of sperm cannot be obtained from all men to
allow insemination of oocytes with the usual numbers of gametes
(100,000 sperm/ oocyte). Initial concerns, that assisted
fertilization with ap-parently defective sperm might lead to the
development of abnormal embryos and an increase in the number
of birth defects, have not been founded. In fact, once fertilization
has been achieved for male factor couples, implantation and
subsequent pregnancy appear to be just as likely, if not more
likely, to occur than in other cases of IVF (18).
Unless advanced age of the female partner is present, IVF is
usually indicated after specific treatment of male and fe-male
factors affecting fertility has been unsuccessful and less invasive
forms of assisted reproduction (intrauterine insem-inations) have
been attempted. If severe male factor infer-tility is present, direct
treatment with IVF and micromanip-ulation may be indicated.
The technique of IVF is described in greater detail elsewhere
(19). Briefly, it involves down-regulation of the woman's pituitary
function with GnRH agonists given during the preceding luteal
phase. This is followed by controlled ovarian hyperstimulation
using FSH or FSH-stimulating agents, to increase the number of
oocytes produced. Follicle development in the ovary is evaluated
directly with transvaginal ultrasound imaging of follicular growth
and by measurement of serial serum estrogen and progesterone
levels. Final oocyte maturation is induced with an intramuscular
dose of hCG (5±10,000 units) when optimal follicular
development is obtained. Retrieval of oocytes is performed by
transvaginal follicular aspiration using ultra-sound guidance with
intravenous sedation. The transvaginal approach has obviated the
need for general anesthesia and laparoscopy to perform IVF.
Many oocytes (mean of 12 oo-cytes) can be obtained from
otherwise normal women with ovarian hyperstimulation.
Morphologically mature, meta-phase II oocytes may then be
inseminated with sperm. Hu-man oocytes survive freezing poorly
since they are in meta-phase; therefore, all retrieved and mature
oocytes are inseminated. Immature oocytes may be matured in
vitro and subsequently inseminated, although only anecdotal
preg-nancies have been achieved after in vitro oocyte maturation.
Sperm are washed free of seminal fluid and inseminated with
oocytes at a concentration of 100,000 or more sperm per oocyte
in simulated human (Fallopian) tubal fluid medium, and the
oocytes that fertilize (embryos) are usually allowed to divide up
to the 8-cell stage before embryo transfer. Em-bryo transfer back
to the uterus is typically performed after 2±3 days of incubation
in vitro. Up to four embryos may be transferred to the uterus, and
excess embryos may be frozen.
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SCHLEGEL AND GIRARDI JCE & M † 1997
Vol 82 † No 3
An implantation rate can be calculated by dividing the num-ber of
gestations (fetal heart on ultrasound) that result from embryo
transfer by the number of total number of embryos transferred to
the uterus in a population of treated patients. Implantation rates
per transferred embryo range from 10 ± 25% in most IVF
programs.
Micromanipulation
Gamete micromanipulation has enabled the embryologist to
circumvent inefficient steps in the fertilization process. Instead of
simply bringing sperm and oocyte together in vitro (IVF),
micromanipulation involves mechanical alteration of the oocyte
in vitro to increase the chance of fertilization of the oocyte by
sperm (Fig. 1.) The three categories of assisted fertilization by
gamete micromanipulation that have been applied in humans are
illustrated in Fig. 2. The first category involves the creation of an
opening in the zona pellucida, an acellular layer surrounding the
oocyte that serves as a major barrier to sperm penetration.
Subsequently, the microma-nipulated oocyte is inseminated
according to standard IVF guidelines. These procedures have
been broadly termed ªzona drilling.º One variant of zona drilling
involving me-chanical piercing of the zona pellucida has been
successful in male factor patients (20). This method has been
called partial zona dissection (PZD; Fig. 2A). A second category
of micro-manipulation techniques directed at facilitating sperm-
oo-cyte interaction is the subzonal insertion of sperm (SuZI).
SuZI involves direct placement of sperm into the perivitelline
space between the zona pellucida and oocyte, completely
bypassing the zona pellucida (Fig. 2B) (2, 21). The third and most
invasive form of microsurgical fertilization is the mi-croinjection
of a single sperm into the cytoplasm of the oo-cyte, referred to as
intracytoplasmic sperm injection (ICSI; Fig. 2C). This technique
for manipulation has a higher risk of oocyte injury than SuZI or
PZD, but overall higher fertili-zation and pregnancy rates (22).
Most importantly, only very few sperm are necessary for ICSI.
The tremendous superi-ority of fertilization and pregnancy rates
after application of ICSI when compared with PZD and SuZI
have relegated both PZD and SuZI to techniques of historical
importance only.
FIG. 1. The structural components of an oocyte important for micro-
manipulation are schematically illustrated.
 CLINICAL REVIEW
FIG. 2. A, Schematic illustration of partial zona dissection technique. B,
Schematic illustration of subzonal insertion procedure. C, The
intracytoplasmic sperm injection technique is schematically pre-sented.
With micromanipulation, fertilization and pregnancy rates appear
to be independent of sperm quality (23, 24), which is the opposite
of what has been demonstrated for both IUI and IVF (16).
Intracytoplasmic sperm injection
Until recently, the clinical application of direct injection of a
single sperm into the cytoplasm of an oocyte during IVF had not
been feasible. The demonstration of fertilization and live births by
Palermo et al. (25) in 1993 was the first suc-cessful application of
ICSI. Since that time, ICSI has been performed extensively in
multiple centers to treat patients with severe male factor
infertility. To date, the success of ICSI procedures has been
related to several factors: 1) the viability of the spermatozoon, 2)
the quality of the oocyte, 3) effective activation of the oocyte, and
4) ability of the oocyte to tolerate intracytoplasmic manipulation.
Application of this treatment is described below.
Indications for ICSI
To date, rigorous indications for ICSI are not universally
agreed upon. In general, any condition in which it is expected that
oocyte fertilization might be impaired, ICSI should be considered.
Early clinical series applied ICSI in cases where men had less
than 500,000 motile sperm present in the ejac-ulate, less than 4%
normal sperm forms (with strict criteria
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711
evaluation), or where couples have failed to fertilize any oocytes
in an earlier IVF cycle. Sperm function tests (2) may provide
additional insight into specific sperm-oocyte inter-action defects
that will define appropriate candidates for ICSI. We have
proposed the following minimal indications for
micromanipulation (26):
a. sperm concentration , 2 3 106 sperm/cc.
b. sperm motility , 5%
c. strict criteria normal morphology , 4% d. use
of surgically retrieved spermatozoa
e. failure of fertilization in a previous IVF cycle Although
fertilization and pregnancy rates with ICSI are
similar to or better than those achieved with normal sperm in
other couples undergoing IVF at the same center (27), couples
with only minor semen abnormalities have not been routinely
treated with ICSI. Given the relatively brief history of ICSI, and
its potential effects on progeny, it would seem prudent to avoid
over-application of this new technology. Therefore, ICSI should
not be recommended to couples for whom there is no documented
benefit, as unknown risk to the embryo and resulting fetus may
still exist (28).
Technique of ICSI
Oocyte processing. Oocytes are prepared by removing the
cumulus mass and corona radiata with hyaluronidase. In-
tracytoplasmic sperm injection is performed on all meta-phase II
oocytes. Metaphase II oocytes have their diploid complement of
chromosomes delicately arranged on the metaphase plate near the
polar body. Mechanical disruption of the metaphase plate can
occur by injury from the injection pipette or by the presence of a
motile sperm in the oocyte cytoplasm. The oocyte is stabilized
with a holding micropi-pette and injected under an inverted
microscope.
Microinjection
Details of the preparation of microtools and protocols for ICSI
are described in detail elsewhere (27). Individual single sperm are
aspirated from a prepared semen specimen and directly injected
into an oocyte immobilized in a droplet of medium under paraffin
oil. The polar body is held at the 12 or 6 o'clock position, and the
injection micropipette contain-ing the single sperm is pushed
through the zona pellucida and oolemma into the cytoplasm of the
oocyte at the 3 o'clock position. Further handling of injected
oocytes is similar to that for oocytes in standard IVF.
Results of ICSI
One of the largest series reporting results using ICSI was from
Van Steirteghem et al. (22) at The Brussels Free Uni-versity in
Brussels, Belgium. In their preliminary report on 150 couples
who underwent 150 consecutive treatment cy-cles, 1409 oocytes
were injected and 830 were successfully fertilized for a
fertilization rate of 59 percent. A total clinical pregnancy rate of
35 percent was achieved. Fertilization and pregnancy rates from
their updated series (29) are shown in Table 1.
In another large series, Palermo et al. (27) reported on 227
couples treated with ICSI for failed IVF cycles or for severe
712 SCHLEGEL AND GIRARDI JCE & M † 1997
Vol 82 † No 3

TABLE 1. Fertilization and clinical pregnancy rates from IVF/ICSI

No. Ongoing or delivered

No. No. Oocyte Fertilization rate Clinical pregnancy
Reference oocytes pregnancy
couples cycles loss (%) per oocyte (%) rate per cycle (%)
injected rate/cycle (%)
Van Steirteghem et al. (66) 1,816 18,778 1,997 (11) 11,629/18,778 (62) 666/1,816 (37)
Palermo et al. (27) 227 227 1,923 136 (7) 1,142/1,923 (59) 94/227 (41) 84/227 (37)
Sherins et al. (31) 190 229 1,690 237 (14) 617/1,690 (60) 38/229 (17) 35/198 (18)
Harari et al. (67) 114 119 1,185 112 (9) 717/1,185 (67) 36/119 (30) 24/119 (20)
Payne et al. (68) 100 100 1,003 672/1,003 (67) 25/100 (25)
Oehninger et al. (30) 92 102 1,163 154 (13) 708/1,163 (61) 31/102 (30) 26/102 (25)
Tsirigotis et al. (69) 69 69 789 84 (11) 410/789 (52) 23/69 (33) 19/69 (28)

male factor infertility. Fertilization and pregnancy rates were
evaluated relative to semen parameters and the origin of the
semen samples. They reported successful fertilization in
1,142/1,923 (59 percent) metaphase II oocytes injected and
ongoing pregnancies in 84/227 (37 percent) couples.
Factors affecting results of ICSI
Spermatozoal factors. Nagy et al. (29) reviewed the effect of
spermatozoal factors on results of ICSI in 966 microinjection
cycles. Despite no normal forms in a semen preparation, virtual
azoospermia or essentially no motile sperm in the ejaculate,
pregnancy could still be achieved. Nagy et al. found that the only
absolute criterion for successful ICSI is the presence of at least
one viable spermatozoon to inject per oocyte in the prepared
pellet of the washed semen sample. The only category of semen
parameters that had a signifi-cantly adverse effect on fertilization
and pregnancy rates with ICSI was when there were no motile
sperm (29). If no motility is present, then viability is often
impaired as well.
Female factors. Oehninger et al. (30) investigated the role of
female factors on ICSI results in a total of 92 couples, where 1163
oocytes were injected, with an overall fertilization rate of 61
percent. Fertilization rates were unaffected by maternal age, but
pregnancy rates were significantly lower with in-creased maternal
age. Pregnancy rates were 49, 23, and 6 percent for couples in
whom maternal age was less than 34 yr, 35±39 yr, and 40 yr or
over. Similar results were found by Sherins et al. (31), with a
30% pregnancy rate for the youngest couples and a 13%
pregnancy rate for the couples with the oldest female partners.
The rate of aneuploidy increased dramatically for embryos
derived from the oocytes of women over 40 compared with those
from women less than 35 yr (32). Implantation of an aneuploid
embryo is highly unlikely. These observations suggest that the
chance of a metaphase
II oocyte being fertilized with ICSI is unrelated to female age, but
the chance of a pregnancy occurring after transfer of ICSI
embryos dramatically decreases with increased female age,
especially female age over 40 yr.
Oocyte activation. Oocyte activation refers to the series of events
that occur after sperm-oocyte fusion during natural fertilization,
which result in the ability of the oocyte to com-plete its nuclear
maturation, to synthesize proteins and DNA. Because sperm
fusion with the oocyte is bypassed during ICSI, other approaches
to induce oocyte activation have been attempted. Tesarik and
Sousa (33) improved fertilization and pregnancy rates during
ICSI by aggressive aspiration and injection of the oocyte
cytoplasm during injection of sperm
into the oocyte to induce oocyte activation. Vigorous cyto-
plasmic aspiration resulted in an increase in fertilization rates per
oocyte from 38 ± 80% when compared with results achieved
using only gentle aspiration of the oocyte cyto-plasm. Pregnancy
rates increased up to 52% with aggressive aspiration/injection.
Aggressive aspiration of cytoplasm caused additional peaks of
oocyte intracellular calcium lev-els, when compared with gentle
aspiration (33). Intracellular calcium changes have long been
thought to have a role in oocyte activation, and these changes
may constitute the mechanism by which aggressive cytoplasmic
aspiration im-proves fertilization rates.
A sperm factor may also have a role in oocyte activation. This
cytoplasmic sperm factor may need to diffuse through the sperm
plasma membrane to induce post-ICSI events in the oocyte that
facilitate pronuclear formation, including for-mation of the sperm
aster by the paternally-derived centro-some and mitosis of the
embryo. Aggressive immobilization of spermatozoa involves
mechanical crushing of the sperm tail between the injection
micropipette and the bottom of the petri dish containing the
spermatozoon. Gerris et al. (34) reported an increase in the
percentage of normally fertilized oocytes from 36 ± 60% with
aggressive immobilization. Pal-ermo et al. (35) found the effect
of aggressive sperm immo-bilization on fertilization rates was
seen primarily in imma-ture spermatozoa that were surgically
retrieved from the epididymis and testis. For epididymal sperm,
Palermo et al. demonstrated an increase in fertilization rates, from
51± 84% per oocyte, with an associated improvement in
pregnancy rates from 51± 82% (35).
It appears that aggressive immobilization of immature
spermatozoa may increase sperm membrane permeability, which
enhances release of cytosolic sperm factors that facil-itate oocyte
activation (36). Alternatively, it is possible that the increased
sperm membrane permeability results in leak-age of toxic factors,
such as reactive oxygen species, out of the cytoplasmic droplet of
immature spermatozoa. Oocyte acti-vation must be induced for
optimal success with ICSI. Cy-toplasmic sperm factors (37) as
well as mechanical stimula-tion of the oocyte are helpful in
inducing oocyte activation.
Cytoplasmic injection/oocyte injury. Disruption of the oocyte
sufficient to cause oocyte demise may occur during ICSI. Results
from some of the major centers performing ICSI show rates of
oocyte loss after injection of 7±14%. Although the precise
reasons for oocyte injury are not known, it is thought to occur as a
result of plasma membrane and ultrastructural disturbances
associated with injection, damage to the meiotic spindle during
injection, and/or extrusion of the oocyte cy-

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 CLINICAL REVIEW 713

toplasm following injection. In addition, other factors such as
changes in temperature have been reported to cause irre-versible
changes in the meiotic spindle of the human oocyte. Clearly, there
is a learning curve for embryologists perform-ing the ICSI
procedure. As greater expertise is gained over the first 50 ±100
oocyte injections, the oocyte injury rate de-creases (38).
Palermo et al. (39) have described an oocyte membrane
response of ªsudden breakageº during attempted ICSI. The
oocytes with this response did not form a normal funnel during
attempted penetration of the injection pipette, but suddenly
separated, spilling the oocyte cytoplasm. With sud-den breakage,
a 14% oocyte injury rate was seen, compared with a 4% injury
rate for other oocytes. Oocytes demonstrat-ing sudden breakage
were more likely to be retrieved from women treated with higher
gonadotropin treatment doses, with lower serum estradiol levels
at retrieval, or where the oocytes were immature, requiring
maturation in vitro. These observations suggest that ovarian
hyperstimulation may af-fect the ability of oocytes to survive
ICSI (39).
Risks of ICSI
Risks of ICSI include general risks of IVF as well as the
specific risks related to the micromanipulation procedure of ICSI.
One of the most significant risks associated with stim-ulation of
the ovaries is the ovarian hyperstimulation syn-drome (OHSS).
This can manifest as massive ovarian en-largement, peritoneal
irritation caused by follicular rupture or hemorrhage, ovarian
torsion, ascites, pleural effusion, ol-iguria, electrolyte imbalance,
hypercoagulability (40), and sometimes death (41). The syndrome
occurs in a moderate form for 3± 4% percent of initiated cycles,
and in a severe form for 0.1± 0.2% of the population (42)
undergoing con-trolled ovarian hyperstimulation. Other reported
complica-tions of ovarian hyperstimulation are pituitary
hemorrhage, endometriotic bloody ascites, and genital cancer
(43).
Complications of ovarian retrieval have been reported for
transvaginal aspiration as well as laparoscopic aspiration.
Complications associated with transvaginal aspiration have been
reported in 0.3±3% of cases and include bleeding, pelvic
infections, and abdominal viscera perforation (44). Laparo-scopic
complications include hemorrhage, intestinal perfo-ration,
infection, and carbon dioxide embolism. The laparo-scopic risks
are no higher in ovarian retrieval procedures than in other
laparoscopic applications.
Finally, pregnancies resulting from ovarian stimulation are at
risk for spontaneous abortion (45), ectopic pregnancy (46), and
multiple gestations (47± 49). The rate of spontane-ous abortion
after achieving a biochemical pregnancy with assisted
reproduction is approximately 25%. These losses are attributed to
a) advanced maternal age and the associated increased prevalence
of chromosomal abnormalities; b) a higher rate of pregnancy loss
resulting from multiple ges-tations, and c) early recognition of
these pregnancies because of close monitoring. After achieving a
clinical pregnancy (the presence of at least one fetal heart beat on
ultrasound), the chance of a spontaneous abortion occurring for
ICSI cycles ranges from 10 ±16%. Ectopic pregnancies occur in
up to 3±5.5% of gestational cycles and can be life threatening.
The
etiology is usually pelvic adhesions and tubal damage from pelvic
inflammatory disease or previous surgery (46). Mul-tifetal
pregnancies occur in 22% of cases of IVF with embryo transfer
(47), and 44 ± 46% of ICSI cases (27, 30) in the United States.
Multifetal pregnancies are considered a complication of assisted
reproductive techniques because of the associated increased
incidence of preeclampsia, placenta previa, pla-cental abruption,
premature rupture of membranes, and postpartum hemorrhage
(48). Most importantly, multiple gestations are almost universally
associated with prematu-rity and the associated complications to
offspring, including cerebral palsy and intracranial hemorrhage
with mental re-tardation or blindness. To prevent multifetal
pregnancies and their attendant complications, it would be
preferable to avoid assisted reproduction unless it is specifically
indicated and to limit the number of embryos transferred. Where
there is government regulation of IVF, including England,
Australia, and France, transfer of only three or fewer em-bryos is
allowed, and multifetal pregnancies are less com-mon (49).
Unfortunately, there is significant pressure to transfer more than
three embryos by couples in the United States who are desperate
to conceive. In general, for women less than 35 yr of age, only
three embryos should be transferred.
Birth defects after assisted reproduction
The bypass of natural barriers to fertilization, possible genetic
defects in men with severe male infertility, and the use of
severely abnormal sperm for intracytoplasmic sperm injection has
engendered concern over the impact of ICSI on the genetic
complement of the offspring (28). Previous stud-ies have
suggested no increase in birth defect rates when IVF alone was
used to induce conception (50). Van Steirteghem
(51) reported no increase in the congenital malformation rate in
their center after ICSI when compared with the general
population. Of 877 children born after ICSI procedures, 23 (2.6
percent) had major congenital malformations compared with 2.0
±2.8% in the general population and 1.9 ±2.9% of children
resulting from IVF without ICSI (51).
Sex chromosome abnormalities have also been reported in
ICSI cases. In't Veld et al. (52) reported on 12 patients with ICSI
pregnancies who underwent prenatal diagnosis for ad-vanced
maternal age. Three of the 12 women had twin preg-nancies for a
total of 15 diagnostic procedures by amniocen-tesis or chorionic
villus sampling. A total of 5 chromosomal abnormalities were
detected: 2 cases of 47 XXY, 1 complex mosaic
45,X/46,X.dic(Y)(q11)/46,X.del(Y)(q11), and 2 cases of 45 XO.
This high rate of sex chromosome abnormalities has not been
corroborated by other studies. The Brussels group reported on a
total of 585 prenatal diagnoses performed in pregnancies
established by ICSI. A total of 6 sex chromosome abnormalities
(1.0 percent) were detected compared with 0.2 percent in the
general population (53). This difference did not achieve statistical
significance. Govaerts et al. (54) reported on 55 karyotypes
obtained by amniocentesis or chorionic villus sampling in
pregnancies from ICSI and found no sex chromosome
abnormalities. When sex chromosome abnor-malities have been
identified it has been unclear whether they were related to the
ICSI procedure, underlying paternal

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714
cytogenetic defects, or advanced maternal age. What is re-
assuring is that the rates of non-sex chromosomal abnormal-ities
in the ICSI population published to date do not exceed the rates
seen in the general population.
The source of sex chromosomal abnormalities in offspring
after ICSI may be related to abnormalities in testicular germ cells.
In addition to disomic sex chromosome abnormalities, several
investigators have reported that up to 13% of men with
azoospermia or severe oligospermia may have deletions of 15,000
±200,000 base pair lengths of the Y chromosome (3± 6). At least
one gene (DAZ; deleted in azoospermia) is deleted in 13% of
patients with nonobstructive azoospermia and some men with
severe oligospermia. Of greater concern is the possibility that
additional unknown genetic problems may be present in infertile
men who have never been able to conceive in the past, but can
now become fathers with ICSI. Evaluation of 32 father-son pairs
after ICSI showed that 3 (9%) ICSI-derived sons had
microdeletions of the DAZ re-gion in one study (6), possibly
because of germ line mosa-icism for Y chromosome
microdeletions in the fathers of 2 of these boys. The third father
had a Y chromosome microde-letion detected on peripheral
leukocyte evaluation that was inherited by his son. ICSI-derived
sons may be at increased risk of infertility or other abnormalities
because of transmis-sion of the genetic defects that are associated
with male infertility.
Although chromosomal abnormality rates in offspring af-ter
assisted reproductive procedures have not exceeded those in the
general population, experience with these tech-niques is brief.
Genetic counseling, preimplantation genetic diagnosis, and state
of the art prenatal diagnosis must also be available to couples
enrolled in assisted reproductive pro-grams. All couples
undergoing micromanipulation proce-dures are strongly urged to
have prenatal diagnosis with amniocentesis or chorionic villus
sampling. The need for prenatal diagnosis is dependent on
whether the couple would consider terminating the pregnancy if
the results are abnormal. If the couple would carry a pregnancy to
term regardless of the results of prenatal diagnosis, then the pro-
cedure of prenatal intervention would carry risks to the fetus
without benefit and therefore cannot be required.
Application of ICSI: epididymal and testicular sperm
The application of ICSI has allowed treatment of couples who
until very recently were considered sterile and untreat-able. Men
with bilateral congenital absence of the vas de-ferens and other
unreconstructable obstructions of the male reproductive tract are
good candidates for ICSI. In these men, microsurgical retrieval as
well as cryopreservation of sperm is possible despite the fact that
the sperm are immature (i.e. have not traversed most of the
excurrent duct system.) Per-cutaneous aspiration of sperm from
the epididymis or testis can also provide sperm for ICSI cycles,
although the sperm are often not of adequate quality for
cryopreservation; there-fore a repeat sperm retrieval procedure
mighty be needed with each ICSI attempt. Using ICSI and
simultaneous open surgical sperm retrieval, clinical pregnancy
rates per sperm and oocyte retrieval attempt range from 45± 82%
at estab-lished centers (35, 55, 56).
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SCHLEGEL AND GIRARDI JCE & M † 1997
Vol 82 † No 3
For men with nonobstructive azoospermia, sperm re-trieval
from the testis is often successful using an open biopsy technique.
Even if a small diagnostic testis biopsy reveals a Sertoli cell-only
pattern in a man with small volume testes and an elevated FSH
level, sperm retrieval might still be possible with a more
extensive biopsy. Taken together, 50 ± 70% of men with
nonobstructive azoospermia can have sperm retrieved surgically
from the testis, with a biopsy performed simultaneous to an IVF-
ICSI procedure (57±59). Up to 20% or more of attempts at sperm
retrieval-ICSI for nonobstructive azoospermia will result in a
clinical preg-nancy (57±59).
Even for men who have no sperm production in their testes,
immature germ cells (spermatids or spermatocytes) might have
application in micromanipulation procedures with some chance of
contributing to pregnancies. In an an-imal model, Kimura and
Yanigamachi (60) have demon-strated induction of pregnancies
by injecting the nucleus of a secondary spermatocyte from a
normal animal into an oocyte and activating the egg with an
electrical pulse. Trans-fer of resulting embryos to the uterus of a
recipient animal resulted in normal offspring. In humans, round
spermatids retrieved from the semen of seven men were
microinjected into oocytes of their female partners, with ongoing
pregnan-cies for two couples, including delivery of a normal child
for one couple (61). The potential for future application of these
techniques is difficult to predict. In our experience, we have
rarely found spermatids in testicular biopsy specimens, un-less
more mature spermatozoa are present.
Associated procedures
Micromanipulation procedures can also be used to analyze and
select embryos with specific genetic, chromosomal, or
biochemical characteristics before the transfer of those em-bryos.
Analysis of the embryo is performed at the four- or eight-cell
stage by extracting an individual cell (blastomere) for evaluation.
Chromosome-specific sequences can be iden-tified using
fluorescent hybridization probes or, alterna-tively, polymerase
chain reaction (PCR) amplification of in-dividual alleles on the
chromosomes themselves may be applied to identify the genotype
of the biopsied embryo. These techniques can allow sex selection
to avoid transmis-sion of X-linked diseases such as hemophilia A
or von Willebrand's disease. In addition, specific genetic defects
such as the homozygous DF508 mutation of the CFTR gene,
associated with the development of a severe form of cystic
fibrosis, can also be identified. These techniques have been
applied for couples known to be at high risk of having chil-dren
with specific genetic diseases. Biopsied embryos have been
successfully transferred, resulting in pregnancies and live births
(62). These micromanipulation techniques are highly labor
intensive and carry some potential pitfalls. For example, if both
male and female partners are heterozygous for the DF508 CFTR
mutation, then an individual embryo has a one-in-four chance of
being homozygous for that gene mutation. However, differential
amplification of either the normal or mutated allele may result in
a false positive or negative result by preimplantation diagnosis
(63).
For sex chromosome analysis, this evaluation is more ac-
 CLINICAL REVIEW
curately performed (up to 95.5% efficiency) using different
colored fluorescent probes (64). A test for both X- and Y-specific
sequences is possible and provides further confir-mation of the
results of these tests. Given the extensive man-power needed for
single-day biopsy and evaluation of the results of embryo biopsy,
this technique is limited to those cases where life threatening
genetic defects can be reliably detected before embryo transfer to
prevent the potential ter-mination of a fetus later in development.
In addition, chro-mosomal mosaicism is common in embryos,
limiting the accuracy of single blastomere biopsy to
approximately 80%.
Summary
Since the first U.S. report of a successful delivery from in vitro
fertilization in 1982 (65), progress in the field of assisted
reproduction and micromanipulation has been truly dra-matic.
Perhaps the most exciting advances have been in the area of male
factor infertility. Couples who previously would have been
offered donor insemination or adoption are now achieving
pregnancies despite severe impairments in semen quality, the
presence of only single numbers of sperm in the ejaculate, or
unreconstructable reproductive tract obstruc-tion. Techniques of
micromanipulation that were revolution-ary less than five yr ago
are now obsolete, replaced by even more successful methods.
Even nonobstructive azoospermia resulting from maturation
arrest or other impairments in germ cell development have been
added to the list of treat-able factors in male infertility, as sperm
can frequently be extracted directly from testicular parenchyma
that is aspi-rated or surgically biopsied. For patients without
sperm in the testicular parenchyma, round spermatid or secondary
spermatocyte injections are at least theoretically possible.
Several important questions remain with regard to IVF-ICSI. 1)
What should be the specific indications for IVF and IVF-ICSI?
Should IVF alone ever be used for male factor infertility? 2) What
are the reasons for failure to achieve pregnancy after ICSI, which
still represent over half of our attempts at achieving ongoing
pregnancies? 3) Can we be certain that using severely impaired or
less mature sperm will not result in significant birth defects or in
genetic ab-normalities that could affect the offspring in
adolescence or adulthood? 4) What is the most successful and
cost effective approach for the infertile couple with impaired
semen parameters?
For couples with male factor infertility, careful evaluation and
treatment of the man should be considered before as-sisted
reproduction, including ICSI. Contemporary applica-tion of ICSI
for severe male factor infertility can allow preg-nancy rates up to
52% (33), with ongoing pregnancy and live delivery rates as high
as 37% per IVF cycle attempt (27). As long as viable sperm are
present in the ejaculate or retrievable from the reproductive tract,
then ICSI procedures can be applied.
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